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The effects of HSV-1 amplicon and polyethyleneimine (PEI)-mediated transfection of dominant negative FGF receptor-1 mutant FGFR1(TK-) into the rat brain substantia nigra (SN) were examined in vivo to model the reduced FGF signaling documented to occur in Parkinson's disease. The number of SN neurons that expressed tyrosine hydroxylase (TH) was significantly reduced following HSV-1 FGFR1(TK-) intranigral delivery and similar changes were observed after PEI-mediated FGFR1(TK-) transfections. Further, we also observed a significantly lower striatal dopamine content following the PEI transfection of FGFR1(TK-). Thus, we conclude that reduced FGF signaling in the SN of Parkinsonian patients could play a role in the impaired dopaminergic transmission associated with the degenerative disease.

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells. We have identified a novel regulatory sequence in the upstream region of the bovine TH gene promoter formed by a dyad symmetry element (DSE1;-352/-307 bp). DSE1 supports TH promoter activity in TH-expressing bovine adrenal medulla chromaffin (BAMC) cells and inhibits promoter activity in non-expressing TE671 cells. DNase I footprinting of relaxed TH promoter DNA showed weak binding of nuclear BAMC cell proteins to a short sequence in the right DSE1 arm. In BAMC cells, deletion of the right arm markedly reduced the expression of luciferase from the TH promoter. However, deletion of the left DSE1 arm or its reversed orientation (RevL) also inactivated the TH promoter. In supercoiled TH promoter, DSE1 assumes a cruciform-like conformation i.e., it binds cruciform-specific 2D3 antibody, and S1 nuclease-cleavage and OsO4-modification assays have identified an imperfect cruciform extruded by the DSE1. DNase I footprinting of supercoiled plasmid showed that cruciformed DSE1 is targeted by nuclear proteins more efficiently than the linear duplex isomer and that the protected site encompasses the left arm and center of DSE1. Our results suggest that the disruption of intrastrand base-pairing preventing cruciform formation and protein binding to DSE1 is responsible for its inactivation in DSE1 mutants. DSE1 cruciform may act as a target site for activator (BAMC cells) and repressor (TE671) proteins. Its extrusion emerges as a novel mechanism that controls cell-specific promoter activity.

The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bone adrenal medullary cells. Angiotensin II-responsive elements are located within -54/+25-bp and -269/-55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12-O-tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.

Activation of the tyrosine hydroxylase (TH) gene in the adrenal medulla during stress is mediated by trans-synaptic mechanisms and may involve cholinergic receptors. Stimulation of nicotinic receptors in adrenal medullary cells induces cell depolarization, influx of Ca2+ ions and increases levels of cAMP. We have shown that both cAMP and membrane depolarization produce an increase in the expression of the TH gene in cultured bovine adrenal medullary cells (BAMC). Others have proposed that transcriptional activation of the TH gene by cAMP is mediated through the sequence homologous to a cAMP responsive element (CRE) located in the proximal region of the TH gene promoter. In the present study we have examined the mechanisms by which membrane depolarization increases the TH gene activity. Treatment of serum-free BAMC cultures with the depolarizing agent, veratridine, increased the extracellular concentration of catecholamines, Met5-enkephalin, and the relative abundance of TH mRNA. Veratridine treatment also increased the levels of mRNAs for the catecholamine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT), and proenkephalin A (PEK). Treatment for longer than 3 h was required to increase TH mRNA levels. By contrast, our previous studies indicated that cAMP stimulation for 2 h produces a maximal increase in TH mRNA levels in BAMC. The effects of veratridine and forskolin on TH mRNA levels were additive, further indicating that depolarization and cAMP activate TH gene expression via different pathways. Calmidazolium, an antagonist of calmodulin, had no effect on the veratridine-induced increase in TH mRNA levels. Similarly sphingosine treatment or preincubation with PMA, which reduce protein kinase C (PKC) activity and attenuate the induction of TH mRNA by PMA or the hormone, angiotensin II, did not affect the induction by veratridine. To identify promoter mechanisms of TH gene activation in depolarized cells we transfected BAMC with a plasmid pTHgoodLuc and treated with veratridine for 24 h. pTHgoodLUC contains a luciferase reporter gene linked to a -428/+21 bp fragment of the bovine TH gene promoter (relative to the transcription start site). Veratridine increased the expression of luciferase from the TH promoter 2.5-fold. Deletion of the -194/-54 bp promoter region containing SP-1 and POU/Oct sites reduced veratridine stimulation by 40%. Additional deletion of the -269 to -190 bp promoter segment, including an AP-1 element, further reduced veratridine stimulation to a statistically non-significant level. In conclusion, activation of TH gene expression upon depolarization is not mediated by calmodulin and PKC. Promoter sequences involved in this activation are located upstream from the CRE. Depolarization may activate TH gene transcription by acting on more than one regulatory region.

The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.

bFGF is a neurotrophic protein expressed in various regions of the adult peripheral and central nervous system. The present study was undertaken to examine the role of bFGF in multihormonal, catecholaminergic and enkephalinergic cells of the adrenal medulla (AM). Western blot analysis revealed the presence of at least three bFGF isoforms (18, 22/23, and 24 kDa) in cultured bovine AM cells. Incubation of AM cells with the exogenous 18 kDa bFGF produced time-dependent increases in tyrosine hydroxylase (TH) and proenkephalin (PEK) mRNA, with maximal changes occurring at 12 h (TH) or 24 h (PEK) of bFGF exposure. Effects of bFGF on TH and PEK mRNA were non-additive with increases induced by exposure of AM cells to nicotine, the depolarizing agent veratridine, or the adenylate cyclase activator forskolin. These data indicate that bFGF effects may occur through intracellular pathways accessed during transsynaptic induction of TH and PEK genes. The increases in PEK mRNA induced by nicotine or bFGF were inhibited by the calcium antagonist TMB-8. TMB-8 also inhibited bFGF-induced increases in TH mRNA as well. However, treatment with TMB-8 increased basal levels of TH mRNA. The addition of bFGF increased endogenous levels of c-fos mRNA, c-Fos and c-Fos-related proteins, suggesting that bFGF may activate TH and PEK gene expression through a calcium-AP1 transcriptional regulatory pathway. Immunohistochemical analysis revealed the presence of bFGF-immunoreactivity (bFGF-IR) in the cytoplasm and in the nucleus of AM cells. Incubation of cells with exogenous bFGF produced time-dependent increases of nuclear bFGF-IR.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary cultures of bovine adrenal medullary cells (AM) in a chemically defined media were used to examine the role of neural and hormonal factors in the expression of proenkephalin A (pEK), phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) genes. Acetylcholine or nicotine reduced cellular content of catecholamines by 30% and increased the relative abundance of pEK, TH, and PNMT mRNAs. The increases produced by acetylcholine were +129%, +147%, and +43% for pEK, TH, and PNMT mRNA, respectively. The kinetics of increases produced by nicotine were different for the 3 mRNAs, with pEK and TH showing enhanced levels over 48 h incubation, while PNMT showed increase during the initial 18 h (+90%) followed by decline to control levels at 48 h. 8-Br cAMP and forskolin elicited a similar pattern of changes as nicotine, suggesting that cyclic AMP may be involved in the mediation of the nicotinic effects. To examine the role of depletion of cellular catecholamines in the regulation of mRNA levels, cells were exposed to tetrabenazine or reserpine. Decreases in cellular catecholamine contents were accompanied by increases in TH and pEK mRNA levels, while the expression of PNMT gene exhibited a transient 4-fold increase and then profound inhibition (60-95%) over a 48-h period. The tetrabenazine effect on TH and pEK mRNA was reduced by alpha-amanitin, suggesting transcriptionally-mediated regulation. Inductions of pEK but not TH or PNMT mRNAs were inhibited by cycloheximide. Hormonal regulation of TH, PNMT, and pEK mRNAs was examined by incubation of cells with dexamethasone. Low concentrations of dexamethasone (0.1, 10 nM) were effective to increase PNMT (+35%, +90%) and pEK (+27%, 45%) mRNA levels. TH mRNA was not affected by similar concentrations of dexamethasone, however, there was a 45% increase at 1 microM. Dexamethasone-elicited increases in PNMT mRNA levels were observed at 48 h and persisted up to 7 days, suggesting that hormonal mechanisms may be distinct from those mediating effects of nicotine, cAMP or tetrabenazine. Taken together, these results indicate that (1) the level of TH, PNMT, and pEK mRNAs are regulated by direct neural (acetylcholine) and hormonal (glucocorticoid) inputs to adrenal medullary cells; (2) effects of acetylcholine could be mediated by cyclic AMP and alterations in catecholamine content; and (3) expression of individual genes is regulated differentially. Such differential regulation of TH, PNMT, and pEK mRNAs may contribute to the long-term selective control of hormonal output from adrenomedullary cells.

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.

The pituitary-adrenocortical axis plays a complex role in the regulation of the levels of enzymes of the catecholamine biosynthetic pathway. In this report we have explored molecular mechanisms of these regulations, by examining the effects of hypophysectomy (HPX) and dexamethasone (DEX) on tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels in the adrenal medulla (AM) and superior cervical ganglia (SCG). Three weeks after hypophysectomy weights (-48%), total RNA (-49%), and DNA (-22%) contents in AM were significantly reduced, when compared to sham-operated animals (SO). In SCG decreases in weight (-23%) and in the ratio of RNA/DNA (-25%) were also found. TH mRNA contents paralleled decreases in total RNA levels and no significant change in the relative abundance of TH mRNA was found. When HPX rats were injected for 5 days with DEX (1 mg/kg, i.p.), TH mRNA levels in the SCG (+51%) and in the AM (+74%) were significantly increased when compared to saline-treated HPX animals. DEX given to SO rats increased TH mRNA in SCG (+49%); a 27% increase in TH mRNA in the AM was also observed. The relative abundance of PNMT mRNA in the AM was reduced after hypophysectomy (-64%). This decrease was completely reversed by DEX. In contrast, DEX did not affect PNMT mRNA levels in the AM of SO rats. PNMT mRNA was not detected in SCG of saline- or DEX-treated rats. In conclusion, our findings suggest that the pituitary-adrenocortical axis is involved in the regulation of the steady-state levels of TH and PNMT mRNAs. This regulation involves: (1) induction of TH mRNA contents in AM and SCG by increased plasma glucocorticoid levels; and (2) maintenance of the steady-state levels of PNMT mRNA in AM by glucocorticoid-dependent mechanisms.

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.

Parkinson's disease usually involves a lengthy preclinical period during which few neurological symptoms are observed despite extensive damage to the dopaminergic nigrostriatal bundle. Injury to this projection in the rat also fails to produce major neurological dysfunctions. In our studies, damage to the nigrostriatal bundle of the rat, resulting in the loss of up to 95% of the dopaminergic terminals in striatum, was accompanied by apparent increases in the synthesis and release of dopamine (DA) from those dopaminergic terminals that remained. More specifically, both the activity of the rate-limiting biosynthetic enzyme, tyrosine hydroxylase, and the content of the principal DA metabolite, dihydroxyphenylacetic acid, were increased in striatum relative to DA levels. The increases were exponentially related to DA loss.